Stereoselective bioconversion of aliphatic dinitriles into cyano carboxylic acids

ABSTRACT

The present invention is directed to a regio- and stereoselective bioconversion of selected aliphatic dinitriles into corresponding cyanocarboxylic acids. More particularly, the present invention provides methods for the conversion of 2-isobutyl-succinonitrile into (S)-3 cyano-5-methylhexanoic acid, which is a useful intermediate in the synthesis of (S)-3(aminomethyl)-5-methylhexanoic acid (pregabalin). Pregabalin can be used for treating certain cerebral diseases, for example, in the treatment and prevention of seizure disorders, pain, and psychotic disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a §371 National Stage filing of PCT/IB05/000873,filed Apr. 1, 2005, which claims the benefit of U.S. ProvisionalApplication 60/562,133, filed Apr. 14, 2004, the entire contents ofwhich are incorporated by reference.

FIELD OF THE INVENTION

The present invention is directed to novel biocatalytic processes forthe regio- and stereoselective conversion of selected aliphaticdinitriles into corresponding cyanocarboxylic acids. More particularly,the present invention provides methods for the conversion of2-isobutyl-succinonitrile into (S)-3-cyano-5-methylhexanoic acid, whichis a useful intermediate in the synthesis of(S)-3-(aminomethyl)-5-methylhexanoic acid (pregabalin). Pregabalin canbe used for treating certain cerebral diseases, for example, in thetreatment and prevention of seizure disorders, pain, and psychoticdisorders. Since pregabalin is effective in improving cerebralfunctions, it is also useful in the treatment of geriatric patients.

BACKGROUND OF THE INVENTION

The enzymatic hydrolysis of organic nitrites to corresponding carboxylicacids and amides provides an important alternative synthetic method to abroad spectrum of useful compounds. Conventional chemical hydrolysis ofnitrites to the corresponding carboxylic acids and amides is typicallycarried out using a strong acid or base catalyst at high reactiontemperatures making it incompatible with compounds which containsensitive functional groups. Furthermore, the poor selectivity ofchemical hydrolysis may result in unwanted by-products along with largequantities of inorganic salts. In contrast, enzymatic nitrile hydrolysisoccurs under mild conditions (neutral pH, 30° C.) offering the potentialfor high chemo-, regio-, and stereoselectivity. As an added advantage,the formation of by-product inorganic salts is avoided.

The best-known industrial applications of nitrile-converting enzymes arethe production of acrylamide (T. Nagasawa et al., Tibtech., 1992, vol.10, 402-408) and nicotinamide (T. Nagasawa et al., Appl. Environ.Microbiol., 1998, vol 54, 1766-1769), using a nitrile hydratase fromRhodococcus rhodochrous J1. Several recent reviews (L. Martinková etal., Current Organic Chemistry, 2003, vol. 7, 1279-1295 and D. Cowan etal., Extremophiles, 1998, vol. 2, 207-216) describe the biochemistry andpotential industrial applications of nitrile converting enzymes.

Enzymatic nitrile hydrolyses are catalyzed by nitrilases, which convertnitrites to the corresponding carboxylic acids, and nitrile hydratases,which convert nitrites to the corresponding amides. Amidases, whichhydrolyze amides to the corresponding carboxylic acids, can be used incombination with nitrile hydratases to convert nitrites to carboxylicacids.

The use of a nitrilase enzyme to prepare a carboxylic acid from thecorresponding nitrile is disclosed in WO 02/072856. Incorporation of theenzyme into a polymer matrix with cross-linking provided a catalyst withimproved physical and biochemical integrity.

The regioselective preparation of ω-nitrilecarboxylic acids fromaliphatic ∝, ω-dinitriles with a biocatalyst was disclosed in U.S. Pat.No. 5,814,508. For example, a catalyst having nitrilase activity wasused to convert 2-methylglutaronitrile into 4-cyanopentanoic acid.

K. Yamamoto, et al. J. Ferment Bioengineering, 1992, vol. 73, 125-129describes the use of microbial cells having both nitrile hydratase andamidase activity to convert trans 1,4-dicyanocyclohexane totrans-4-cyanocyclohexanecarboxylic acid.

Regioselective biocatalytic conversions of dinitriles to cyanosubstituted carboxylic acids, have been reported for a series ofaliphatic α, ω-dinitrile compounds using microbial cells having analiphatic nitrilase activity or a combination of nitrile hydratase andamidase activities (J. E. Gavagan et al. J. Org. Chem., 1998, vol. 63,4792-4801).

Stereoselective enzymatic conversions of nitriles have been describedfor the preparation of chiral carboxylic acids and amides enriched inone enantiomer (M Wieser et al., Chapter in StereoselectiveBiocatalysis, Marcel Dekker Inc.: New York, 2000, 461-486). Astereoselective nitrilase enzyme from Alcaligenes faecalis ATCC 8750 isused to prepare (R)-mandelic acid from racemic mandelonitrile (K.Yamamoto et al., Appl. Environ. Microbiol., 1991, vol. 57, 3028-3032). Anitrilase from Rhodococcus rhodochrous NCIMB 11216 preferentiallyhydrolyzes (+)-2-methylhexanitrile in a racemic mixture of2-methylhexanitrile leaving (−)-2-methylhexanitrile unreacted (M.Gradley et al., Biotechnology Lett., 1994, vol. 16, 41-46). U.S. Pat.No. 5,593,871 disclosed a process for preparing 2-alkanoic acid amidesenriched in one enantiomer, from nitriles using microorganismscontaining stereoselective nitrile hydratases. Enantiopure α-amino acidsand amides were prepared from racemic α-aryl and α-alkyl-substitutedglycine nitriles using Rhodococcus sp. AJ270 containing astereoselective nitrile hydratase and a stereoselective amidase (M.-C.Wang et al., J. Org. Chem., 2002, vol. 67, 6542). The foregoingreferences are hereby incorporated herein in their entirety.

The therapeutic value of racemic pregabalin, particularly its efficacyas an anticonvulsant, has been found to be attributable primarily to the(S)-enantiomer. Toward the goal of providing cost-effective pregabalindrug therapy, a number of synthetic routes to the (S)-enantiomerenriched compound have been investigated. For example, asymmetrichydrogenation of the appropriate cyano substituted olefin followed byreduction of the cyano group to the corresponding amine providespregabalin substantially enriched in the (S) enantiomer (United StatesPatent Application Publication No. 2003/0212290).

The synthesis of pregabalin, its derivatives and analogs by purelychemical methods is disclosed in U.S. Pat. Nos. 6,642,398; 6,635,673;and 6,046,353.

SUMMARY OF THE INVENTION

In the process of the present invention, regio- and stereoselectivebiocatalytic conversions of aliphatic dinitriles to cyanocarboxylicacids are achieved using enzyme catalysts having nitrilase activity.

The present invention relates to a novel method for preparing an(S)-enantiomer of a compound of formula I:

wherein C3 has an (S) configuration;

R¹ is hydrogen, (C₁-C₆) alkyl or phenyl; and

R² is (C₁-C₈) alkyl, (C₂-C₈) alkenyl, (C₃-C₈) cycloalkyl, —O(C₁-C₆)alkyl, —CH₂—CH₂—O— (C₁-C₆)alkyl, (C₁-C₆)alkyl-OH,-phenyl-(C₁-C₆)alkyl-OH, -phenyl-O—(C₁-C₆)alkyl, phenyl or substitutedphenyl;

with the proviso that when R² is methyl, R¹ is hydrogen, (C₁-C₆) alkylor phenyl;

comprising the steps of:

(1a) contacting a compound of formula II:

with an enzyme catalyst having nitrilase activity in a reaction medium;and

(1b) recovering the (S)-isomer of the compound of formula I from thereaction medium; and, optionally recovering unchanged (R)-isomer ofcompound II.

Compounds of formula I are useful in synthesizing compounds havingpharmaceutical activity, such as pregabalin.

In a preferred embodiment of the invention, R¹ and R² are independentlyhydrogen or C₁ to C₃ alkyl.

In a preferred embodiment of the invention, the compound of formula IIis a racemic mixture comprising 3R and 3S isomers.

A preferred embodiment of the present invention is the process wherebyracemic 2-isobutyl-succinonitrile (the compound of formula II wherein R¹is H and R² is methyl) is converted into (S)-3-cyano-5-methylhexanoicacid (the compound of formula I wherein R¹ is H and R² is methyl)comprising the steps of:

(2a) contacting racemic 2-isobutyl-succinonitrile with an enzymecatalyst having nitrilase activity in a reaction medium; and

(2b) recovering (S)-3-cyano-5-methylhexanoic acid from the aqueousmixture; and, optionally recovering unchanged(R)-2-isobutylsuccinonitrile.

Preferably the reaction medium is an aqueous medium.

In a preferred embodiment of the present invention, the recovered andunchanged (R)-isomer of compound II is subsequently racemized by heatingwith a weak base in the presence of an organic solvent. A preferred baseis 1,8-diazabicyclo[5.4.0.]undec-7-ene and a preferred solvent istoluene. Optionally the resulting racemate of II may be recycled intoeither of the above stated processes at step (1a) or (2a).

In one embodiment of the present invention the enzyme catalyst is in theform of whole microbial cells, extracts of microbial cells, partiallypurified enzymes, purified enzymes or enzyme catalysts that areimmobilized on a support.

In another embodiment of the present invention, the enzyme catalyst is apartially purified enzyme. Examples of partially purified enzymesinclude, but are not limited to NIT-101, NIT-102, NIT-103 (BioCatalyticsInc., Pasadena, Calif.), and nitrilase from Arabidopsis thaliana (JülichFine Chemicals, Jülich, Germany).

In a preferred embodiment of the present invention the nitrilase enzymecatalyst is immobilized on a support. Examples of immobilized nitrilaseenzyme catalysts include but are not limited to NIT-102 C2(BioCatalytics Inc., Pasadena, Calif.), NIT-102 immobilized on Eupergit(Röhm GmbH & Co. KG, Darmstadt, Germany), and nitrilase from Arabidopsisthaliana immobilized on Eupergit. In a preferred embodiment theimmobilized nitrilase enzyme catalyst is NIT-102 C2.

In another embodiment, the reaction media is comprised of distilledwater or buffered water. Preferably the buffered water is buffered to apH in the range of about 5.0 to about 10.0 and most preferably to a pHin the range of about 6.0 to about 8.0.

The present invention also relates to a process for the preparation of(S)-3-(aminomethyl)-5-methylhexanoic acid (pregabalin) comprising thesteps of:

(a) contacting racemic 2-isobutyl-succinonitrile with an enzyme catalysthaving nitrilase activity in a reaction medium;

(b) recovering (S)-3-cyano-5-methylhexanoic acid from the reactionmedium;

(c) converting (S)-3-cyano-5-methylhexanoic acid into an acid salt; and

(d) hydrogenating the acid salt to form(S)-3-(aminomethyl)-5-methylhexanoic acid (pregabalin).

Preferably, the acid salt has the formula

wherein M is Na, K, Li, NH₄, NH₂R⁶R⁷,

NH₃R¹ or NH(R⁶)₂R⁷ wherein R⁶ and R⁷ are each independently (C₁-C₆)alkyl.

For convenience, certain terms employed in the specification, examplesand appendant claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

The term “alkyl” is a straight or branched group of from 1 to 8 carbonatoms including but not limited to methyl, ethyl, propyl, butyl,iso-butyl, and tert-butyl.

The term “cycloalkyl” as used herein includes moieties derived fromcyclic hydrocarbons containing from three to seven ring carbon atoms,including cyclic hydrocarbon moieties substituted with straight orbranched alkyl moieties.

The term “alkoxy”, as used herein, means “alkyl-O—”, wherein “alkyl” isdefined as above.

The term “alkenyl” is intended to include hydrocarbon chains of either astraight or branched configuration comprising one or more carbon-carbondouble bonds which may occur in any stable point along the chain, suchas ethenyl and propenyl. Alkenyl groups typically will have 2 to about12 carbon atoms, more typically 2 to about 8 carbon atoms.

The term racemate, as used herein, means an equimolar mixture of a pairof enantiomers. A racemate is usually formed when synthesis results inthe generation of a stereocenter. As used herein, the term racemicmixture means racemate.

As used herein, the term enantiomers refers to compounds which at themolecular level are nonsuperposable with mirror images of each other.Enantiomers may exist in either the (R) or (S) configuration.

As used herein, the term stereoselective synthesis refers to a chemicalreaction that leads to formation of a single stereoisomer or anenantiomer-enriched mixture of isomers from among two or more possiblestereoisomers.

As used herein, the term regioselective refers to a reaction that takesplace at a single atom or group of atoms from among two or more possibleatoms or groups of atoms. The regioselective hydrolysis of a dinitrileresults in the conversion of a single nitrile group to a carboxyl group.

“° C.” means degrees-Celsius;

The term “enzyme catalyst”, as used herein, means a catalyst which ischaracterized by either a nitrilase activity or a combination of anitrile hydratase activity and an amidase activity. The catalyst may bein the form of a whole microbial cell, permeabilized microbial cell(s),one or more cell component of a microbial cell extract, partiallypurified enzyme(s), or purified enzyme(s).

As used herein, the term enantiomer excess refers to the mole fractionof the dominant enantiomer in a mixture of enantiomers expressed as apercentage.

The term “aqueous reaction mixture” means a mixture of the substrate andenzyme catalyst in a largely aqueous medium.

The term “nitrilase activity” means an enzyme activity that converts anitrile group to a carboxylic acid group.

The term “nitrile hydratase activity” as used herein, means an enzymeactivity that converts a nitrile group to an amide group.

The term “amidase activity” means an enzyme activity that converts anamide group to a carboxylic acid group.

ATCC is American Type Culture Collection located at 10801 UniversityBoulevard, Manassas, Va., 20110-2209, U.S.A. BioCatalytics Inc. islocated at 129 N. Hill Avenue, Suite 103, Pasadena, Calif., 91106,U.S.A. Jülich Fine Chemicals GmbH is located at Rudolf-Schulten-Straβe5, D-52428 Jülich, Germany.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an enzymatic method for preparingaliphatic cyanocarboxylic acids of formula I from dinitriles of formulaII. Any suitable method commonly used in the art may be used to preparethe dinitrile (II) starting materials.

Scheme 1 refers to a specific embodiment of the present inventionwherein a chemo-enzymatic method is used in the conversion of2-isobutyl-succinonitrile (V) into (S)-3-cyano-5-methylhexanoic acid(VI). Compound VI may be used as an intermediate in the synthesis ofpregabalin (VII) as illustrated in Scheme 2. Step 3 of Scheme 1 depictsthe racemization of by-product (R)-isomer (Va) and subsequent recycleinto Step 2.

In Step 1 of Scheme 1 racemic 2-isobutyl-succinonitrile (V) is formed bythe condensation of isovaleraldehyde (III) with ethylcyanoacetate (IV)followed by the addition of KCN. The racemate arises from thestereocenter created at the C₃ carbon atom of V.

Step 2 of Scheme 1 depicts the regio- and stereoselective hydrolysis ofthe dinitrile V racemate yielding (S)-3-cyano-5-methylhexanoic acid (VI)plus unchanged (R)-isomer of V.

The nitrilase catalyzed hydrolysis of 2-isobutyl-succinonitrile (V) into(S)-3-cyano-5-methylhexanoic acid VI is both regioselective andstereoselective. Regioselectivity is based upon conversion of the cyanogroup into a carboxyl group at the C1 carbon atom only. The reaction isstereoselective in that the (S) enantiomer of V is predominantlyinvolved in the conversion leaving the (R)-enantiomer essentiallyunchanged.

As illustrated in Scheme 2, an acid salt VIa of S-cyanoacid VI ishydrogenated in a subsequent step to obtain(S)-3-(aminomethyl)-5-methylhexanoic acid (pregabalin). The reaction iscarried out in the presence of a hydrogenation catalyst, preferablyRaney nickel. Acceptable acid salts include compounds of formula VIawherein M is Na, K, Li, NH₄, NH₂R⁶R⁷, NH₃R⁶ or NH(R⁶)₂R⁷ wherein R⁶ andR⁷ are independently (C₁-C₆)alkyl.

In the regio- and stereoselective conversion of racemate V into(S)-cyanoacid VI, as depicted in Scheme 1, the nitrilase enzyme reactspredominantly with the (S) enantiomer. Accordingly the reaction mixtureis increasingly enriched in the (R) enantiomer Va as the conversionprogresses.

Another objective of the current invention is to avoid economic waste byrecycling or reusing the unchanged (R)-dinitrile Va. The presentinvention, therefore, provides a method for the racemization of the(R)-dinitrile (Step 3, Scheme 1) and subsequent recycle through Step 2of Scheme 1.

Various enzymes of the present invention, having nitrilase activity or acombination of nitrile hydratase and amidase activities, can be foundthrough screening protocols such as enrichment isolation techniques,which initially select microorganisms based on their ability to grow inmedia containing the enriched nitrile. Enrichment isolation techniquestypically involve the use of carbon-limited or nitrogen-limited mediasupplemented with an enrichment nitrile, which can be the nitrilesubstrate for the desired bioconversion, or a structurally similarnitrile compound. Microorganisms that possess nitrilase activity can beinitially selected based on their ability to grow in media containingthe enrichment nitrile. Gavagan et al., (Appl. Microbiol. Biotechnol.(1999) vol. 52, 654-659) used enrichment techniques to isolate aGram-negative bacterium, Acidovorax facilis 72W (ATCC 55746), from soil,using 2-ethylsuccinonitrile as the sole nitrogen source. Acidovoraxfacilis 72W (ATCC 55746) was shown to be useful for the selectiveconversion of 2-methylglutaronitrile to 4-cyanopentanoic acid.Enrichment techniques were also used to isolate the thermophilicbacterium, Bacillus pallidus Dac521, which catalyzes the conversion of3-cyanopyridine to nicotinic acid (Almatawah and Cowan, Enzyme Microb.Technol. (1999) vol. 25, 718-724). Microorganisms isolated by enrichmenttechniques can be tested for nitrile hydrolysis activity by contactingsuspensions of microbial cells with a nitrile compound and testing forthe presence of the corresponding carboxylic acid using analyticalmethods such as high performance liquid chromatography, gas liquidchromatography, or liquid chromatography mass spectrometry (LCMS).Techniques for testing the nitrile hydrolysis activity of Acidovoraxfacilis 72W (ATCC 55746) are reported in U.S. Pat. No. 5,814,508.

Once a microorganism having nitrilase activity or nitrile hydratase andamidase activities has been isolated, enzyme engineering can be employedto improve various aspects of the enzyme(s). These improvements can beuseful for the present invention and include increasing selectivity,catalytic efficiency of the enzyme, stability to higher temperatures anda wider range of pH, and enabling the enzyme to operate in a reactionmedium including a mixture of aqueous buffer and organic solvent.

A variety of techniques, which can be employed in the present invention,to produce an enzyme catalyst having nitrilase activity or nitrilehydratase and amidase activities in addition to having an improvedyield, throughput, and product quality suitable for a particularbioconversion process, include but are not limited to enzyme engineeringtechniques such as rational design methods which include site-directedmutagenesis, and directed evolution techniques utilizing randommutagenesis or DNA shuffling techniques.

Suitable enzyme catalysts for the conversion of compounds of formula IIinto compounds of formula I are in the form of whole microbial cells,permeabilized microbial cells, extracts of microbial cells, partiallypurified enzymes or purified enzymes, and such catalysts can beimmobilized on a support.

This process can be carried out in a single phase by contacting2-isobutyl-succinonitrile with an enzyme catalyst in distilled water, orin an aqueous solution of a buffer, which will maintain the initial pHof the reaction between 5.0 and 10.0, preferably between 6.0 and 8.0.Suitable buffering agents include potassium phosphate and calciumacetate. As the reaction proceeds, the pH of the reaction mixture maychange due to the formation of an ammonium salt of the carboxylic acidfrom the corresponding nitrile functionality of the dinitrile. Thereaction can be run with no pH control, or a suitable acid or base canbe added over the course of the reaction to maintain the desired pH.However, as indicated above it is possible to produce enzyme catalystsusing technologies such as enzyme engineering and directed evolution,which will operate effectively over wider pH ranges.

This process can be carried out in reaction mixtures comprised of twophases: an aqueous phase, which initially contains enzyme and dissolved2-isobutyl-succinonitrile, and an organic phase, which consist mainly ofracemic 2-isobutyl-succinonitrile. Two-phase reaction mixtures areprepared by adding 2-isobutyl-succinonitrile to an aqueous solution ofenzyme and buffer agents such that the amount of2-isobutyl-succinonitrile added exceeds it aqueous solubility limit. Theaqueous solubility limit of 2-isobutyl-succinonitrile in 50 mM potassiumphosphate (30° C., pH 7.5) is approximately 0.06M. Over the course ofthe reaction, (S)-3-cyano-5-methylhexanoic acid ammonium salt is formedand increases in concentration in the aqueous phase, while the organicphase decreases in volume and becomes enriched in(R)-2-isobutyl-succinonitrile. Alternately, this process can also becarried out in reaction mixtures comprised of three phases: an aqueousphase, which initially contains dissolved 2-isobutyl-succinonitrile, anorganic phase, which consists mainly of racemic2-isobutyl-succinonitrile, and a solid phase, which consists of enzymeimmobilized on an insoluble support. Three-phase reaction mixtures areprepared by the procedure described for two-phase reaction mixtureexcept that an enzyme immobilized on an insoluble support is used inplace of an un-immobilized enzyme.

Optionally, the enzyme may be immobilized in a polymer matrix or aninsoluble support. Immobilized enzyme catalysts can be used repeatedlyand in continuous processes, and can be separated from the products ofthe enzymatic process more easily than un-immobilized enzyme catalysts.Methods for the immobilization of enzymes in a polymer matrix, such ascalcium alginate or polyacrylamide, or an insoluble support, such ascelite, are well known to those skilled-in-the-art. NIT-102 C2(BioCatalytics Inc., Pasadena, Calif.), which is a nitrilase enzymeimmobilized on an insoluble support, is particularly useful for theconversion of II to III, since it can be used repeatedly in batch orcontinuous processes. The concentration of NIT-102 C2 used in a reactionis chosen to obtain a desired reaction rate and depends on the specificactivity of the catalyst and the concentration of substrate. Typically,NIT-102 C2 is used in the range of about 0.001 g to 0.3 g moist weightper mL of reaction volume, with a preferred range of 0.01 to 0.15 gmoist weight per mL of reaction volume.

Additionally, several lyophilized lysates prepared from microbial cellsand designated as NIT-101, NIT-102, NIT-103 (BioCatalytics Inc.,Pasadena, Calif.), and nitrilase from Arabidopsis thaliana (Jülich FineChemicals, Jülich, Germany) are also useful for the conversion of II toIII. Contact of NIT-101, NIT-102, NIT-103 and A. thaliana nitrilase withI in an aqueous reaction mixture results in the formation of II.Reactions using NIT-101, NIT-102, NIT-103 and nitrilase from Arabidopsisthaliana, can be carried out in two-phase reaction mixtures usingcatalyst concentrations ranging from 0.001-0.04 g dry weight per mlreaction volume, with a preferred range of 0.002-0.02 g dry weight permL reaction volume.

The temperature of the hydrolysis reaction is chosen to both optimizethe reaction rate and the stability of the enzyme catalyst activity. Thetemperature of the reaction may range from just above the freezing pointof the suspension (ca. 0° C.) to 60° C., with a preferred range ofreaction temperature from 5° C. to 35° C.

Recovery of the (3S) isomer of the compound of formula I and recovery ofunchanged (3R) isomer of the compound of formula II may be carried outusing suitable separation, isolation and purification techniqueswell-known to those skilled in the art.

In a preferred method of recovery, the unchanged (3R) isomer of thecompound of formula II is separated from the basic aqueous reactionmixture by extraction with an organic solvent such as ethyl acetate. Theacid salt of the (3S) isomer of the compound of formula I ispreferentially dissolved in the aqueous layer and is subsequentlyisolated by acidification and extraction with an organic solvent such asethyl acetate.

The compounds of formula I can be used to synthesize compounds, such aspregabalin, having usefulness in the treatment of such disorders asepilepsy, convulsion, anxiety, pain, and neurodegenerative disorders,including Alzeimer's disease, Huntington's disease and Parkinson'sdisease.

Examples of specific compounds of formula I are the following compounds:

-   (S)-3-cyano-5-methyl-octanoic acid;-   (S)-3-cyano-5-methyl-heptanoic acid;-   (S)-3-cyano-5-methyl-hexanoic acid;-   (S)-3-cyano-5-methyl-nonanoic acid;-   (S)-3-cyano-5-ethoxy-hexanoic acid;-   (S)-3-cyano-5-cyclohexyl-hexanoic acid; and-   (S)-3-cyano-5-trifluoromethyl-hexanoic acid.

Example 1 Preparation of 2-isobutyl-succinonitrile

A mixture of ethyl cyanoacetate (73.3 g, 6.48 mol), isovaleraldehyde(613.9 g, 7.13 mol), piperidine (5.5 g, 0.065 mol), and hexane (0.5 L)was placed under reflux with continuous removal of water. When noadditional water was collected, the mixture was cooled and distilledunder vacuum to remove solvent. Isopropanol (1 L) was added to theremaining oil, followed by a solution of potassium cyanide (422 g, 6.48mol) in water (2 L). The reaction mixture was maintained below 35° C.during addition of the potassium cyanide solution and then held atapproximately 35° C. for 4 h. The reaction mixture was distilled atatmospheric pressure until a temperature of 95° C. was reached and thenrefluxed at this temperature for 5 h. The reaction mixture was cooled,diluted with water (0.5 L) and extracted with 1 L methyl tert-butylether (MTBE). The MTBE extract was washed with water (0.5 L), dried overanhydrous magnesium sulfate, filtered, and concentrated under vacuum togive 873.4 g of 2-isobutyl-succinonitrile as an oil. Purified samples of2-isobutyl-succinonitrile can be obtained by vacuum distillation (90° C.at 0.275 mm Hg).

¹H NMR (CDCl₃, 400 MHz): δ 0.93-0.99 (m, 6H), 1.43-1.50 (m, 1H),1.71-1.78 (m, 1H), 1.81-1.91 (m, 1H), 2.69 (d, 2H, J=6.5 Hz), 2.90-2.97(m, 1H).

Example 2 Preparation of (S)-3-Cyano-5-methylhexanoic Acid from2-isobutyl-succinonitrile with NIT-101, NIT-102. NIT-103, andArabidopsis thaliana nitrilase

Three 8 mL screw-cap glass vials were each charged with2-isobutyl-succinonitrile (20 mg), 1 mL of 50 mM potassium phosphatebuffer (pH 7.5, 2 mM dithiothreitol (DTT)), and 10 mg of a nitrilaseenzyme selected from NIT-101, NIT-102, or NIT-103 (Biocatalytics Inc.,Pasadena, Calif.). One 8 mL screw-cap glass vial was charged with2-isobutyl-succinonitrile (20 mg) and 1 mL of a solution of Arabidopsisthaliana nitrilase in 50 mM phosphate buffer (pH 7.8) containing 100 mMethylenediaminetetraacetic acid (EDTA) and 2 mM DTT (Jülich FineChemicals, Jülich, Germany). The four reaction mixtures were stirredwith magnetic stir-bars for 15 h at 30° C. and then individuallyextracted with ethyl acetate (2×6 mL). After removing the ethyl acetateextracts, the aqueous parts were treated with 4N HCl (0.15 mL) andextracted with ethyl acetate (3×6 mL). Ethyl acetate extracts of theacidified aqueous parts were concentrated under vacuum to give 7.8 mg(34.2% yield), 8.8 mg (38.6% yield), 8.1 mg (35.5% yield), and 4.0 mg(17.5% yield) of (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA) for thereactions performed with NIT-101, NIT-102, NIT-103, and A. thaliananitrilase, respectively. Samples of (S)-3-cyano-5-methylhexanoic acidfrom each of the reactions were treated with an excess of(trimethylsilyl)diazomethane to give their methyl ester derivatives andanalyzed by gas chromatography (GC) on a Chiraldex™ G-TA column (30M×0.25 mm ID, 125 micron film thickness) to determine enantiomericpurities. The enantiomeric purities of the NIT-101, NIT-102, NIT-103,and A. thaliana nitrilase reaction products were 96.3%, 91.1%, 95.5%,and 98.5% e.e., respectively. (e.e. means “enantiomer excess”)

Example 3 Preparation of (S)-3-Cyano-5-methylhexanoic Acid from2-isobutyl-succinonitrile with NIT-102

A 125 mL jacketed reaction vessel maintained at 30° C. was charged with2-isobutyl-succinonitrile (3.33 g), NIT-102 (0.5 g) and 122 mL of 50 mMpotassium phosphate buffer (pH 7.5) containing 5 mM DTT and 1 mM EDTA(reaction buffer). After stirring for 12.5 h, the product mixture wasextracted with ethyl acetate (4×50 mL). The ethyl acetate extracts wereremoved, and the aqueous part was adjusted to pH 2.5 with 4M HCl andextracted with ethyl acetate (3×50 mL). The ethyl acetate extracts ofthe acidified aqueous part were combined, dried with anhydrous MgSO₄,filtered, and concentrated under vacuum to give 1.56 g of (S)-CMHA(41.1%). A sample of the reaction product was treated with(trimethylsilyl)diazomethane and analyzed by GC as described in example2 to reveal an enantiomeric purity of 98.5% e.e.

¹H NMR (CDCl₃, 400 MHz): δ 0.93-0.97 (m, 6H), 1.30-1.37 (m, 1H),1.61-1.68 (m, 1H), 1.82-1.89 (m, 1H), 2.57-2.63 (m, 1H), 2.72-2.78 (m,1H), 2.98-3.06 (m, 1H).

Example 4 Preparation of Potassium (S)-3-Cyano-5-methylhexanoate from2-Isobutyl-succinonitrile with NIT-102 C2

Two 125 mL jacketed reaction vessels maintained at 30° C. were eachcharged with 2-isobutyl-succinonitrile (6.81 g), NIT-102 C2 (1.70 g) and118.2 mL of reaction buffer. After stirring for 24 h, the productmixtures were decanted, leaving the enzyme catalyst in the reactionvessels. Reaction buffer (20 mL) was added to the each reaction vessel,stirred for approximately 2 min., and then decanted and added to theproduct mixtures. Reactions were repeated by adding2-isobutyl-succinonitrile (6.81 g) and reaction buffer (118.2 mL) toeach reaction vessel and stirring the reaction mixtures for 24 h. Afterfour reactions were completed in each vessel (total of eight batchreactions), the product mixtures were combined and extracted with MTBE(3×500 mL). The MTBE extracts were removed and the aqueous part adjustedto pH 2.1 with phosphoric acid and extracted with MTBE (2×500 mL). TheMTBE extract of the acidified aqueous part was concentrated under vacuumto leave an oil, which was treated with water (100 mL) and KOH (8.5 g).The resulting solution was concentrated under vacuum to give 24.2 g(31.3%) of potassium (S)-3-cyano-5-methylhexanoate. Methyl(S)-3-cyano-5-methylhexanoate was prepared from potassium(S)-3-cyano-5-methylhexanoate and analyzed by chiral GC to reveal anenantiomeric purity of 99.1% e.e.

¹H NMR (D₂O, 400 MHz): δ 0.75-0.78 (m, 6H), 1.18-1.25 (m, 1H), 1.43-1.50(m, 1H), 1.53-1.68 (m, 1H), 2.28-2.38 (d, 2H, J=6.5 Hz), 2.86-2.93 (m,1H).

Example 5 Preparation of (S)-3-Cyano-5-methylhexanoic Acid from2-Isobutyl-succinonitrile with NIT-102 C2 Under Nitrogen Atmosphere

A 125 mL jacketed reaction vessel maintained at 30° C. was charged with2-isobutyl-succinonitrile (6.53 g), NIT-102 C2 (2.61 g), 120 g ofreaction buffer, and purged with nitrogen. The resulting mixture wasstirred for 24 h and then decanted to a 250 mL glass bottle, leaving thecatalyst in the reaction vessel. The reaction was repeated by rechargingthe reaction vessel containing the used catalyst with2-isobutyl-succinonitrile (6.53 g) and 120 g of reaction buffer, purgingwith nitrogen, and stirring the resulting mixture for 24 h. Reactionsamples (0.1 mL) were mixed with 0.4 mL ofwater:methanol:trifluoroacetic acid (60:40:0.09, v/v/v) and analyzed byHPLC on a Symmetry™ C8 column (150×3.9 mm) maintained at 30° C. Thecolumn was eluted with water:methanol:trifluoroacetic acid (60:40:0.09,v/v/v) and detection was carried out with a refractive index detector.

A total of fifty batch reactions were carried out with catalyst recycle.Product mixtures from two consecutive batch reactions were combined andextracted with ethyl acetate (2×150 mL). The aqueous part was thenadjusted to pH 2 with 4M HCl and extracted with ethyl acetate (2×150mL). The ethyl acetate extracts of the acidified aqueous part werecombined, dried over anhydrous magnesium sulfate, filtered, andconcentrated under vacuum to yield (S)-CMHA. A total of 160.8 g (43.2%yield) of (S)-CMHA was obtained from fifty batch reactions. Initialrates for reactions one, twenty-six, and fifty, were 14.8, 17.4, and15.1 mM (S)-CMHA/h, respectively. Chiral GC analysis of the methyl esterderivative of (S)-CMHA isolated from batch reactions 39 to 50 revealedan average enantiomeric purity of 99.0% e.e.

Example 6 Preparation of (S)-3-Cyano-5-methylhexanoic Acid from2-Isobutyl-succinonitrile with NIT-102 C2 Under Ambient Atmosphere

A series of batch reactions for the conversion of2-isobutyl-succinonitrile to (S)-CMHA using NIT-102 C2 was carried outas described in example 5 except that reactions were carried out underambient atmosphere instead of nitrogen atmosphere. Reaction samples wereanalyzed by HPLC as described in example 5.

A total of fifty batch reactions were carried out with catalyst recycleunder ambient atmosphere. Initial reaction rates determined fromreaction samples taken at four hours were 14.2, 13.2 and 9.3 mM(S)-CMHA/h for reactions one, twenty-six, and fifty, respectively.

Example 7 Preparation of tert-Butylammonium(S)-3-Cyano-5-methylhexanoate

Product mixtures from the conversion of 2-isobutyl-succinonitrile to(S)-CMHA (Example 6, reactions 37-44) were combined and extracted withethyl acetate (2×250 mL). The ethyl acetate extracts were dried overanhydrous magnesium sulfate, filtered and concentrated under vacuum togive an oil (32.5 g, 62.2% yield) that was mainly(R)-2-isobutyl-succinonitrile. The aqueous part was adjusted to pH 2with 4M HCl and extracted with ethyl acetate (2×250 mL). The ethylacetate extracts were concentrated to a volume of 470 mL and thenstirred while tert-butylamine (15.9 mL, 151.5 mmol) was added dropwise.The white crystalline salt that formed was collected by filtration andair-dried overnight to give 30.0 g of t-butylammonium(S)-3-cyano-5-methylhexanoate. Methyl (S)-3-cyano-5-methylhexanoate wasprepared from t-butylammonium (S)-3-cyano-5-methylhexanoate and analyzedby chiral GC to reveal an enantiomeric purity of 99.5% e.e.

¹H NMR (CDCl₃, 400 MHz): δ 0.90-0.94 (m, 6H), 1.26-1.32 (m, 10H),1.54-1.61 (m, 1H), 1.78-1.88 (m, 1H), 2.30-2.35 (m. 1H), 2.43-2.50 (m,1H), 2.96-3.04 (m, 1H).

Example 8 Preparation of (S)-3-Aminomethyl-5-Methylhexanoic Acid fromPotassium (S)-3-Cyano-5-Methylhexanoate

A mixture of potassium (S)-3-cyano-5-methylhexanoate (20 g, 103.5 mmol),water (50 mL), 45% KOH (12 g), isopropanol (12 g), and Raney Nickel wereshaken overnight in a Parr Shaker under 50 psi of hydrogen. The mixturewas filtered, heated to approximately 50° C., treated with acetic acid(6.5 mL) and stirred overnight at room temperature. The mixture was thenadjusted to slightly above pH 7 with 45% KOH and concentrated undervacuum to remove most of the isopropanol. Isopropanol (20 mL) was addedto the mixture, which was then acidified with acetic acid, stirredovernight at room temperature, and filtered to give 4.3 g of(S)-3-aminomethyl-5-methylhexanoic acid as a white crystalline solid.The enantiomeric purity was determined to be 100% e.e. by preparing aderivative of (S)-3-aminomethyl-5-methylhexanoic acid using Marfey'sreagent (Na-(2,4-dinitro-5-fluorophenyl)-L-alaninamide) and analyzing byHPLC on a BDS Hypersil C18 column (250×4.6 mm, 5μ) eluted withacetonitrile:1% triethylamine (pH 3) (38:62, v/v).

Example 9 Preparation of (S)-3-Aminomethyl-5-Methylhexanoic Acid fromt-Butylammonium (S)-3-Cyano-5-Methylhexanoate

A mixture of t-butylammonium (S)-3-cyano-5-methylhexanoate (26 g, 113.9mmol), water (48.8 mL), ethanol (35.8 mL), KOH (7.2 g, 91% flake), andSponge Nickel™ (A-7000, 16.3 g water wet, Activated Metals & Chemicals,Inc., Sevierville, Tenn.) was shaken overnight in a Parr Shaker under 50psi of hydrogen. The mixture was filtered (celite) and the cake washedwith water (10 mL) and ethanol (5 mL). Acetic acid (9.4 mL) was added tothe filtrate and the resulting mixture was stirred overnight at 4° C.The product was filtered, rinsed with 10 mL of isopropyl alcohol, anddried under vacuum to give 11.1 g (61%) of a white solid. A portion(10.0 g) of this material was crystallized from a 1:1 mixture ofisopropyl alcohol and water to give 8.8 g of(S)-3-aminomethyl-5-methylhexanoic acid in 100% ee.

Example 10 Racemization of (R)-2-Isobutyl-succinonitrile Using DBU

The racemization of (R)-2-isobutyl-succinonitrile was carried out onmaterial recovered from bioconversion of racemic2-isobutyl-succinonitrile with NIT-102 C2. A mixture of(R)-2-isobutyl-succinonitrile (1.36 g, 10 mmol, 69% ee), toluene (5 mL)and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 0.076 g, 5 mmol) wasrefluxed for 2 h. Water (10 mL) was added to the reaction and theresulting mixture extracted with ethyl acetate (2×10 mL). The combinedorganic extracts were washed sequentially with 5% HCl (20 mL) andsaturated aqueous sodium chloride (20 mL), dried over anhydrousmagnesium sulfate, filtered, and concentrated under vacuum to giveracemic 2-isobutyl-succinonitrile (1.14 g, 84%). Enantiomeric purity wasdetermined by GC using a Chiraldex™ G-TA column (30 M×0.25 mm ID, 125micron film thickness).

Example 11 Racemization of (R)-2-Isobutyl-succinonitrile UsingAmberlite® IRA-400

Amberlite® IRA-400 resin (1 g wet weight, Rohm & Haas, Philadelphia,Pa.) was stirred with 5% NaOH (10 mL) for 10 minutes and washed withwater until the washings were neutral. Ethanol (25 mL) and(R)-2-isobutyl-succinonitrile (69% ee) were added to the resin and theresulting mixture refluxed for 2 h. The reaction mixture was filteredand concentrated under vacuum. The residue was taken up into ethylacetate (25 mL) and washed with water (3×100 mL). The organic phase wasdried over anhydrous magnesium sulfate, filtered, and concentrated undervacuum to give racemic 2-isobutyl-succinonitrile (0.81 g, 81%).

1. A process for preparing (S)-3-(aminomethyl)-5-methylhexanoic acid(pregabalin) comprising the steps of: (a) contacting2-isobutyl-succinonitrile with an enzyme catalyst having nitrilaseactivity in a reaction medium; (b) recovering(S)-3-cyano-5-methylhexanoic acid from the reaction medium; (c)converting (S)-3-cyano-5-methylhexanoic acid into an acid salt; and (d)hydrogenating the acid salt to form (S)-3-(aminomethyl)-5-methylhexanoicacid (pregabalin).
 2. The process according to claim 1, whereinunchanged (R)-3-cyano-5-methylhexanoic acid is recovered from thereaction medium of step (a).
 3. The process according to claim 1 whereinsaid unchanged (R)-3-cyano-5-methylhexanoic acid of step (a) isracemized by heating with base in the presence of an organic solvent toform racemic 2-isobutyl-succinonitrile and step (a) is repeated usingsaid racemic 2-isobutyl-succinonitrile.
 4. The method of claim 1 whereinsaid enzyme catalyst is a nitrilase in the form of whole microbialcells, permeabilized microbial cells, extracts of microbial cells,partially purified enzymes, purified enzymes or an enzyme catalystimmobilized on a support.
 5. A method according to claim 1 wherein saidenzyme catalyst is selected from the group consisting of NIT-101,NIT-102, NIT-103 and nitrilase from Arabidopsis thaliana.